Epstein-Barr virus (EBV) is associated with a number of malignancies of B lymphocyte and epithelial origin. Many of these are specific to or increased in AIDS patients including epithelial cancers of the head and neck. This proposal focuses on the development of an in vitro model of EBV infection of epithelium using organotypic cultures. Organotypic cultures differ from monolayers in that the cells differentiate and stratify to resemble epithelium found in situ. Because EBV can replicate in differentiated epithelial cells in vivo, organotypic culture is an ideal system to study the role of epithelial cells in the viral life cycle. Our preliminary data demonstrate that we can efficiently infect primary oral keratinocytes in culture using a recombinant EBV that expresses GFP as a marker, and that we can generate organotypic cultures of primary oral keratinocytes that effectively mimic oral mucosal epithelium. Based on these preliminary data, we propose to characterize EBV infection in organotypic culture identifying the cells infected and the viral gene products expressed. Several studies have suggested that EBV may establish a latent infection in vivo. We will determine whether EBV can establish a latent infection and characterize the type of latency. The functions of the viral gene products expressed in epithelium will be examined. Thus, we will establish and characterize a model system for the study of EBV in epithelium and use this system to define the functions of the viral gene products as far as possible within the limited scope of the R21 funding mechanism. These studies will set the stage for future experiments that will examine several long standing questions concerning EBV infection of human oral epithelium. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is associated with a variety of human malignancies that occur predominantly in B lymphocytes or epithelial cells, many of which have an increased incidence in AIDS patients. The proposed research is intended to develop an in vitro model of EBV infection of epithelium that will be invaluable in filling in the gaps in our knowledge about the role of epithelial cells in the EBV lifecycle. Because the oral mucosa is the site of entry and exit of the virus, epithelial cells play a major role in viral spread and thus, understanding their role in the viral life cycle will not only shed light on this important step but has the potential to impact all downstream events from EBV infection. This model is likely to be important not only in adding to our knowledge of the role of the epithelium in the viral life cycle and the contribution of the virus to the development of malignancies, but could also be used for the development and/or testing of antiviral strategies.